Preparation of (R)-3-Hydroxybutyric Acid or Its Salts by One-Step Fermentation

ABSTRACT

The subject invention relates to a genetically modified microorganism comprising a (R)-3-hydroxybutyric acid pathway and being able of producing (R)-3-hydroxybutyric acid, and a method of preparing (R)-3-hydroxybutyric acid or a salt thereof using the genetically modified microorganism.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of and claims priority to U.S. application Ser. No. 16/542,044, filed on Aug. 15, 2019, which is a divisional application of U.S. application Ser. No. 15/944,331, filed on Apr. 3, 2018, which claims priority to U.S. application No. 62/481,476, filed on Apr. 4, 2017, the contents of which are incorporated herein by reference in their entirety.

TECHNICAL FIELD OF THE INVENTION

The subject invention pertains to the field of bioengineering, in particular, genetically modified microorganisms having (R)-3-hydroxybutyrate biosynthesis capability, and a method for preparing (R)-3-hydroxybutyric acid and its salts by microbial fermentation.

BACKGROUND OF THE INVENTION

(R)-3-hydroxybutyrate (R-3HB) is an optically chiral compound with the CAS No. 625-72-9. Naturally occurring (R)-3-hydroxybutyric acid is produced by the metabolism of long chain fatty acids in the liver of mammals. It exists as a major ketone in plasma and peripheral tissues and can be used as an energy source in most tissues of the body.

(R)-3-hydroxybutyric acid has positive effect on treating many diseases and nutritional functions as well. For example, it can be used to treat many diseases that arise from elevated levels of ketone (such as nerve disorders including epilepsy and myoclonus, and neurodegenerative diseases including Alzheimer's disease and dementia); it can reduce free radical damage by oxidizing the coenzyme Q (such as ischemia); it can enhance the efficiency of metabolism to achieve the treatment of inadequate support, angina, myocardial infarction, etc. by improving training efficiency and athletic performance; it can also be used to treat cancer related diseases such as brain cancer (astrocytoma, etc.). Further, it has good effects on the treatment of glucose metabolism disorders (such as type-1 diabetes, type-2 diabetes, hypoglycemia ketone disease, etc.). It can be used to control osteopenia (osteopenia), osteoporosis, severe osteoporosis and related fractures. Based on these functions and therapeutic and nutritional effects, (R)-3-hydroxybutyric acid and its salts can be used as food additives and drugs with great health and medicinal values.

(R)-3-hydroxybutyric acid has been prepared primarily by chemical methods. It can be made from direct chemical synthesis or prepared by enzymatic degradation of poly-3-hydroxybutyrate with poly-3-hydroxybutyric acid depolymerase. The chemical synthesis of (R)-3-hydroxybutyric acid requires a high temperature, a high pressure and expensive chiral metal catalysts. The process of enzymatic degradation of poly-3-hydroxybutyric acid requires a large amount of organic solvent, and very pure poly-3-hydroxybutyrate as starting material. Besides the long reaction time, it is difficult to control the racemization of product after reaction. Moreover, this method needs more optimization in the laboratory to meet even higher requirements for industrial scale commercialization because of high cost and low efficiency.

At present, most of the commercially available 3-hydroxybutyric acid is a racemic mixture, with the ratio of (R)-3-hydroxybutyric acid to (S)-3-hydroxybutyric acid being about one to one. Although study showed that (S)-3-hydroxybutyric acid is not physiologically active, racemic 3-hydroxybutyric acid and its salts, especially sodium salts, are still the main commodity form and accepted by most consumers. It is expected that a single optical (R)-3-hydroxybutyric acid and its salts will become popular in future, replacing the racemic 3-hydroxybutyric acid and its salts.

Generally, it is believed that naturally or biologically produced compounds are safer (and non-toxic) than chemically produced compounds. People prefer the “natural” or “biological” feature of the source of pharmaceutical, food and cosmetic ingredients.

BRIEF SUMMARY OF THE INVENTION

In order to produce “safe and nontoxic” (R)-3-hydroxybutyric acid by a biosynthetic method, the inventors of this invention have developed genetically modified nonpathogenic microorganisms and unexpectedly found that fermentation with such genetically modified nonpathogenic microorganisms in an unusually efficient one-step process can produce (R)-3-hydroxybutyric acid with high yield and high purity. Optimally engineered organisms have been screened and selected by genetic engineering technology to enhance the expression of genes associated with (R)-3-hydroxybutyric acid main metabolic pathway and weaken the branched metabolic pathway. After that, (R)-3-hydroxybutyric acid can be effectively accumulated in the fermentation process, which has broad application prospect in industrial scale.

Accordingly, in one aspect, the invention provides a genetically modified microorganism having (R)-3-hydroxybutyrate biosynthesis capability, comprising one or more exogenous nucleic acids encoding at least one (R)-3-hydroxybutyric acid pathway enzyme selected from the group consisting of succinyl-CoA transferase, acetoacetyl-CoA synthase, and 3-HB dehydrogenase.

The genetically modified microorganism is able to produce (R)-3-hydroxybutyric acid by a novel (R)-3-hydroxybutyric acid pathway which includes the following steps:

-   -   (i) converting pyruvic acid and coenzyme A to acetyl-CoA,     -   (ii) converting acetyl-CoA into acetoacetyl-CoA,     -   (iii) converting acetoacetyl-CoA to acetoacetic acid, and     -   (iv) converting acetoacetic acid to (R)-3-hydroxybutyric acid.         The genetically modified microorganism comprises one or more         exogenous nucleic acid encoding at least one         (R)-3-hydroxybutyric acid pathway enzyme expressed in a         sufficient amount to produce (R)-3-hydroxybutyric acid. The         overexpressed pathway enzyme is selected from succinyl-CoA         transferase, acetoacetyl-CoA synthase, and 3-HB dehydrogenase.

In some embodiments, the exogenous nucleic acid is a codon-optimized gene for better expression in the genetically modified microorganism.

In some embodiments, the overexpressed pathway enzyme is selected from the group consisting of succinyl-CoA transferase, acetoacetyl-CoA synthase, and 3-HB dehydrogenase.

In some embodiments, the exogenous nucleic acid encoding succinyl-CoA transferase is at least 90% identical to the nucleotide sequence of SEQ ID NO:1, the exogenous nucleic acid encoding acetoacetyl-CoA synthase is at least 90% identical to the nucleotide sequence of SEQ ID NO:2, and the exogenous nucleic acid encoding 3-HB dehydrogenase is at least 90% identical to the nucleotide sequence of SEQ ID NO:3.

In some embodiments, the genetically modified microorganism is a nonpathogenic microorganism. Examples of suitable nonpathogenic microorganism includes, but not limited to, Corynebacterium glutamicum, Bacillus subtilis, Brevibacterium lactofermentum, Brevibacterium difficile, Brevibacterium flavum, or Brevibacterium breve.

In some preferred embodiments, the nonpathogenic microorganism is Corynebacterium glutamicum and Bacillus subtilis. In some more preferred embodiments, the microorganism is Corynebacterium glutamicum (such as the strain deposited at the China General Microbiological Culture Collection Center under the accession number CGMCC No. 13111).

In another aspect, this invention provides a biological method for efficiently producing (R)-3-hydroxybutyric acid using the genetically modified microorganism. In some embodiments, the method is a one-step fermentation process. In some preferred embodiments, the genetically modified nonpathogenic microorganism is capable of producing (R)-3-hydroxybutyric acid in a one-step fermentation process of this invention.

Specifically, the present invention provides a process for producing (R)-3-hydroxybutyric acid by fermenting with a genetical modified nonpathogenic microorganism in a fermentation broth. In this process, the fermentation broth comprises carbon and nitrogen sources and at least one enzyme that is overexpressed by the nonpathogenic microorganism. The carbon and nitrogen sources are directly converted into (R)-3-hydroxybutyric acid by one-step fermentation with the nonpathogenic microorganism, and the (R)-3-hydroxybutyric acid was recovered from fermentation broth after it excreted into the broth during fermentation process. The nonpathogenic microorganism is selected from a group consisting of Corynebacterium glutamicum, Bacillus subtilis, Brevibacterium lactofermentum, Brevibacterium difficile, Brevibacterium flavum and Brevibacterium breve.

In some embodiments, the carbon source comprises a member selected from the group consisting of glucose, sucrose, maltose, molasses, starch and glycerol. In some other embodiments, the nitrogen source comprises a member selected from the group consisting of an organic nitrogen source and an inorganic nitrogen source. Examples of suitable organic nitrogen source include, but are not limited to, corn steep liquor, bran hydrolyzate, soybean cake hydrolyzate, yeast extract, yeast powder, peptone, and urea; wherein Examples of suitable inorganic nitrogen source include, but are not limited to, ammonium sulfate, ammonium nitrate, and aqueous ammonia.

In some embodiments, the (R)-3-hydroxybutyric acid produced by the process of this invention is free of bacterial endotoxin and has a purity of 95% or more.

In some embodiments, the (R)-3-hydroxybutyric acid prepared by the process of this invention is in the form of (R)-3-hydroxybutyrate sodium salt, (R)-3-hydroxybutyrate potassium salt, (R)-3-hydroxybutyrate magnesium salt, or (R)-3-hydroxybutyrate calcium salt.

In another aspect, this invention provides a food grade (R)-3-hydroxybutyric acid and salts thereof.

In still another aspect, this invention also provides food grade racemic 3-hydroxybutyric acid and its salts to meet the needs of the market. The racemic 3-hydroxybutyric acid is prepared by racemization treatment of (R)-3-hydroxybutyric acid or a (R)-3-hydroxybutyrate salt produced in accordance of the process of this invention.

In yet another aspect, the invention provides a recombinant nucleic acid segment encoding succinyl-CoA transferase, acetoacetyl-CoA synthase, and 3-HB dehydrogenase.

The invention comprises the following technical scheme:

Fermentation of (R)-3-hydroxybutyric acid is carried out by using a genetically modified non-pathogenic microorganism, which directly converts the carbon and nitrogen sources into (R)-3-hydroxybutyric acid which is excreted into the fermentation broth and could be recovered directly. The microorganism can be one or more members selected from the group consisting of Corynebacterium glutamicum, Bacillus subtilis, Brevibacterium lactofermentum, Brevibacterium difficile, Brevibacterium flavum and Brevibacterium breve. These microorganisms have the following biotransformation functions: converting pyruvic acid and coenzyme A to acetyl-CoA; converting acetyl-CoA into acetoacetyl-CoA; converting acetoacetyl-CoA to acetoacetic acid; and then converting acetoacetic acid to (R)-3-hydroxybutyric acid.

Preferably, the microorganism over-expresses one or more enzymes selected from the group consisting of succinyl-CoA transferase, acetoacetyl-CoA synthase, and 3-HB dehydrogenase (3-HB Dehydrogenase, 3-HBDH).

More preferably, the microorganism overexpresses succinyl-CoA transferase, acetoacetyl-CoA synthase and/or 3-HB dehydrogenase.

Different media may be used for different microorganisms in the fermentation process. The carbon source may be selected from the group consisting of glucose, sucrose, maltose, molasses, starch and glycerol. One or more organic or inorganic nitrogen sources may be used in a fermentation medium. The organic nitrogen source may come from the group consisting of corn steep liquor, bran hydrolyzate, soybean cake hydrolyzate, yeast extract, yeast meal, peptone and urea; and the inorganic nitrogen source may include one or more compounds selected from the groups consisting of ammonium sulfate, ammonium nitrate, and aqueous ammonia.

In a preferred embodiment of the present invention, the fermentation medium includes glucose 75 g/L, corn steep liquor 25-30 g/L, (NH₄)₂SO₄ 20 g/L, KH₂PO₄ 1.5 g/L, MgSO₄.7H₂O 0.5 g/L, urea 1.0 g/L, histidine 30 mg/L, molasses 25 g/L, biotin 100 μg/L, and defoamer 0.2 g/L when Corynebacterium glutamicum was applied.

Preferably, the feed medium includes ammonium sulfate 500 g/L and glucose 650 g/L when batch feed fermentation was performed.

The purity of the (R)-3-hydroxybutyric acid prepared by a fermentation method of the present invention could be greater than 95%, greater than 96%, greater than 97%, greater than 98%, or even greater than 99%.

Pure (R)-3-hydroxybutyric acid thus prepared does not contain bacterial endotoxin and chemical odor such as bitterness.

(R)-3-hydroxybutyric acid is an acid which forms a salt with a base. Alternatively, the (R)-3-hydroxybutyric acid prepared by present invention may exist in the form of a salt, such as sodium salt, potassium salt, magnesium salt, or calcium salt, preferably in the form of a sodium salt. These salts may all be optically active compounds.

Since racemic 3-hydroxybutyric acid and its sodium salt have traditionally been accepted by food and pharmaceutical manufacturers and consumers, (R)-3-hydroxybutyric acid and its salts can be subjected to racemic treatment to prepare racemic 3-hydroxybutyric acid and its salts. For example, racemization can be achieved by heating (R)-3-hydroxybutyric acid in an alkaline solution such as sodium hydroxide solution for a certain time.

The racemic 3-hydroxybutyrate salt may be sodium 3-hydroxybutyrate, potassium 3-hydroxybutyrate, magnesium 3-hydroxybutyrate, or calcium 3-hydroxybutyrate.

(R)-3-hydroxybutyric acid and its salts prepared by the present invention are free of bacterial endotoxin and toxic chemicals, therefore ensuring food safety. In addition, (R)-3-hydroxybutyric acid can be used directly to manufacture pharmaceuticals and health products as it contains no chemical residues or chemical reaction impurities, and it does not have chemical odor such as bitterness as well.

The genetically engineered microorganisms constructed in the present invention can effectively produce and accumulate (R)-3-hydroxybutyric acid in the fermentation broth during the fermentation process, and could give rise to food-grade (R) level by downstream process, which has broad industrial prospects.

Corynebacterium glutamicum has been engineered to produce (R)-3-hydroxybutyric acid at high yield. A strain of this microorganism has been deposited under the accession number CGMCC No. 13111 on Oct. 14, 2016, at China General Microbiological Culture Collection Center, located in Institution of Microbiology, Chinese Academy of Sciences, Building 3, No. 1 Beicheng West Road, Chaoyang District, Beijing, China 100101 (www.cgmee.net).

As used herein, the term “one-step fermentation” refers to a fermentation process that includes adding one or more fermenting agents (e.g., a microorganism) to a fermentation medium which is fermented to give the desired product, without having to add a second round of fermenting agent and then going through a second round of fermentation process.

As used herein, the term “nonpathogenic microorganism” refers to a microorganism that generally does not cause disease, harm or death to another microorganism, an organism, or human being.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an exemplary pathway for producing (R)-3-hydroxybutyric acid. The first step is a native pathway of the microorganism, and the other steps are genetically modified pathways, in which enzymes are overexpressed. The overexpressed enzymes catalyzing the pathway reactions are acetoacetyl-CoA synthase, Succinyl-CoA transferase and 3-HB dehydrogenase.

FIG. 2 shows the plasmid map for the vector pXMJ19-t3-OXCT1-BDH1.

FIG. 3 shows the plasmid map for the vector pMA5-t3-OXCT1-BDH1.

FIG. 4 shows an HPLC spectrum showing the production of (R)-3-hydroxybutyric acid from the genetically modified nonpathogenic microorganism (Top: reference, Bottom: sample).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is further described in detail with specific embodiments. The following examples are intended to demonstrate the invention and not to limit the scope of the invention.

Mass percentage is referred in the invention such as the added amount, content and concentration of multiple substances unless otherwise provided descried or defined.

In the embodiments provided under the present invention, room temperature (15-30° C.) is referred to by default unless otherwise provided descried or specified.

Fermentation of (R)-3-hydroxybutyric acid by microorganism was investigated in the present invention in order to supply the consumer and pharmaceutical and food manufacturers with the “naturalized” or “biogenic” sources of (R)-3-hydroxybutyric acid and its salts, 3-hydroxybutyric acid and its salts.

The inventors screened and selected species for the construction of genetic engineering strains. It is not considered that general strain with potentially pathogenic feature such as Escherichia coli. Nonpathogenic microorganism was chosen and genetically engineered as fermentation strains such as Corynebacterium glutamicum, Bacillus subtilis, Brevibacterium lactofermentum, Brevibacterium difficile, Brevibacterium breve and Brevibacterium brevica. Several strains were obtained through screening which are able to produce (R)-3-hydroxybutyric acid by fermentation. No endotoxin was produced at all during fermentation, which may cause harm to most people. So, it is considered as “non-toxic and harmless” design.

It is necessary to adjust and control some parameters such as dissolved oxygen, temperature, pH etc., to have a higher yield of (R)-3-hydroxybutyric acid during fermentation.

The constant dO₂ is controlled at 15% to 30% during fermentation. Fermentation can be carried out under the following conditions: air flow is about 1-1.3 vvm, where vvm is the ratio of the amount of ventilation per minute to the actual volume of liquid in the tank (for example, 1 vvm is equal to 30 L/min for a fermenter containing 30 liters of fermentation broth, and 1 vvm is equal to 5 L/min as for a fermentation tank containing 5 liters of fermentation broth).

Preferably, the temperature is first controlled at 30^(˜)32° C. during the initial stage of fermentation and then increased to 34^(˜)37° C. at the later stage of the fermentation which facilitates the synthesis and excretion of (R)-3-hydroxybutyric acid by the microorganism.

The pH is generally controlled at pH 6.0^(˜)8.0, preferably at pH 6.5^(˜)7.0, during the initial stage of fermentation. It can then be adjusted to 6.8^(˜)7.0 in the later stages of fermentation to facilitate the synthesis and drainage of (R)-3-hydroxybutyric acid from the fermentation broth.

The above term “later stage of fermentation” refers to from exponential stage to stationary stage of microbial growth. For example, the OD_(600 nm) value is no longer rising and tending to decrease when monitoring cell density with OD_(600 nm) values.

The residual sugar is controlled at 1.0%^(˜)3.0% during fermentation process, more precisely at 1.5%^(˜)2.5%.

After the fermentation is completed, the fermentation broth needs to be recovered and (R)-3-hydroxybutyric acid is extracted therefrom. For example, the supernatant of the fermentation broth is obtained by centrifugation. The supernatant is concentrated if necessary; (R)-3-hydroxybutyric acid is separated by a post-treatment such as purification and drying.

Cells and macromolecules in the fermentation medium can be removed by filtration (including ultrafiltration, nanofiltration, etc.). Concentrated filtration, and other post-processing means such as drying, purification and other methods may be applied if necessary to isolate (R)-3-hydroxybutyric acid. Alternatively, (R)-3-hydroxybutyric acid can be isolated by centrifugation which obtains supernatant of fermentation broth, then go through ultrafiltration, nanofiltration and other methods to remove macromolecules, or through concentrated filtration if necessary, then by drying, purification and other post-processing means.

To prepare (R)-3-hydroxybutyrate such as sodium salt, potassium salt, magnesium salt, calcium salt, an equivalent amount of (R)-3-hydroxybutyric acid is reacted with the corresponding base or metal oxide such as sodium hydroxide. The reaction temperature is controlled to be 30° C. or lower, preferably at 25° C. or lower, and more preferably at 20° C. or less, where the racemic reaction could be avoided as much as possible.

As the whole preparation process does not require or involve an organic solvent, chemical odor like bitterness was not detected from the product (R)-3-hydroxybutyric acid which could be directly used to manufacture pharmaceuticals and health care products.

As used herein, the term “microorganism” is intended to mean any organism that exists as a microscopic cell that is included within the domains of archaea, bacteria or eukarya. Therefore, the term is intended to encompass prokaryotic or eukaryotic cells or organisms having a microscopic size and includes bacteria, archaea and eubacteria of all species as well as eukaryotic microorganisms such as yeast and fungi. The term “nonpathogenic microorganism” refers to a microorganism that is not capable of causing disease or harmful responses in a host. In preferred embodiments, the microorganism in this invention is nonpathogenic microorganism selected from the group consisting of Corynebacterium glutamicum, Bacillus subtilis, Brevibacterium lactofermentum, Brevibacterium difficile, Brevibacterium breve and Brevibacterium brevica.

As used herein, the terms “overexpressed”, “overexpression” and the like, refer to the expression of a polypeptide or protein encoded by a DNA introduced into a host cell, wherein the polypeptide or protein is either not normally present in the host cell, or wherein the polypeptide or protein is present in the host cell at a higher level than that normally expressed from the endogenous gene encoding the polypeptide or protein.

As used herein, the term “modified” or “engineered” refers to any manipulation of a microorganism, wherein the manipulation includes but not limited to inserting a polynucleotide and/or polypeptide heterologous to the microorganism and mutating a polynucleotide and/or polypeptide native to the microorganism. The term “genetically modified microorganism” refers to a microorganism having at least one genetic alteration not normally found in the wild type strain of the reference species, for example, involving rational pathway design and assembly of biosynthetic genes, genes associated with operons, and control elements of such polynucleotides, for the production of a desired product.

As used herein, the term “pathway” is “biosynthetic pathway”, which refers to a set of biochemical reactions for converting one chemical species into another.

As used herein, the term “native” or “endogenous” as used herein with reference to molecules, in particular nucleic acids, enzymes and polynucleotides, which are originated or present in the organism. It is understood that expression of native enzymes or polynucleotides may be modified in genetically modified microorganisms.

On the contrary, the term “exogenous” refers to molecules, in particular nucleic acids, enzymes and polynucleotides, which are not originated, but introduced into the host organism. For example, the molecule can be introduced by introduction of an encoding nucleic acid into the host genetic material such as by integration into a host chromosome or as non-chromosomal genetic material such as a plasmid.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLE 1 Genetically Modification of Corynebacterium glutamicum

The genetically modified Corynebacterium glutamicum comprises an exemplary (R)-3-hydroxybutyric acid synthesis pathway. FIG. 1 depicts the exemplary pathway (with the native pathway of the microorganism in the black frame; the engineered pathway, in which enzyme are overexpressed, in the red frame; and the overexpressed enzymes labeled near the red arrows.

Plasmid Construction

Codon optimized genes for t3, OXCT1 and BDH1, encoding acetoacetyl-CoA synthase, succinyl-CoA transferase and 3-HB dehydrogenase respectively, were cloned into plasmid pUC57 to give a cloning vector PUC57-t3-OXCT1-BDH1. The codon optimized nucleic acid sequences are listed in SEQ ID NOs: 1-3 respectively. Table 1 below lists the exemplary pathway enzymes.

TABLE 1 Exemplary pathway enzymes for production of (R)-3-hydroxybutyric acid Gene Name Enzyme Name GenBank ID t3 acetoacetyl-CoA synthase WP_007111820 OXCT1 Succinyl-CoA transferase AAH09001.1 BDH1 3-HB dehydrogenase EAW53609.1

Next, the gene fragment t3-OXCT1-BDH1 was digested from the cloning vector PUC57-t3-OXCT1-BDH1 using restriction endonuclease enzymes EcoR1 and Hind3, and inserted into the same endonuclease digested plasmid pXMJ19 to construct recombinant vectors. For confirming the insertion, the constructed recombinant vectors were transformed into competent bacterial strain DH5a for clone selection. The presence of the inserted fragment t3-OXCT1-BDH1 was confirmed by colony-PCR using specific primers, restriction enzyme digestion and sequencing. The confirmed vector was the expression vector named as pXMJ19-t3-OXCT1-BDH1. FIG. 2 is the plasmid map for pXMJ19-t3-OXCT1-BDH1.

Generation of Genetically Modified Corynebacterium glutamicum

The Corynebacterium glutamicum ATCC 13032 cells were made electro-competent. Then, the expression vector pXMJ19-t3-OXCT1-BDH1 was transformed into this competent cell and used for producing (R)-3-hydroxybutyric acid. The transformed bacteria were plated on 2% agar plate with CM medium (beef extract 3 g/L, peptone 10 g/L, NaCl 5 g/L, and 0.5 g/L D-alanine) containing 25 mg/L chloramphenicol for clone selection.

This modified Corynebacterium glutamicum harboring plasmid pXMJ19-t3-OXCT1-BDH1 is the strain deposited at the China General Microbiological Culture Collection Center under the accession number CGMCC 13111.

EXAMPLE 2 Fermentation of the Genetically Modified Corynebacterium glutamicum

The genetically modified C. glutamicum strain CGMCC No. 13111 was take out from a glycerol stock stored at −80° C. and was used to inoculate a 5000 mL flask containing 500 mL seed medium (75 g/L of glucose, 25-30 g/L of corn steep liquor, 20 g/L of (NH₄)₂SO₄, 1.5 g/L of KH₂PO₄, 0.5 g/L of MgSO₄.7H₂O, 1.0 g/L of urea, 30 mg/L of histidine, 25 g/L of molasses, 100 μg/L of biotin, pH 7.0). The cells were cultured at 30° C. for 18 hours, at which time the absorbance at 600 nm (known as OD₆₀₀) reaches 0.4-0.5.

Next, the 500 mL seed culture was inoculated into a 7-liter fermenter filled with 5 liters fermentation medium, which was the same as the seed medium described above. The pH of the medium was controlled at 6.4^(˜)6.7 after autoclaving. The fermentation began with an agitation speed of 600 rpm and aeration rate of 1 vvm. The tank pressure was maintained at 0.05 Mpa, and the cultivation temperature was controlled at 30° C. IPTG was added with a final concentration of 0.1 mmol/L after 8-10 h.

At the later stage of the fermentation, the pH was controlled at 6.7 and the temperature was raised to 35° C. The dissolved oxygen concentration (dO₂) was controlled at 15^(˜)25% by adjusting aeration rate and agitation speed. To maintain the residual sugar level, sugar was fed slowly while the concentration of the original sugar dropped to about 3.0%. The residual sugar level was maintained at 1.5%^(˜)2.0%.

After 72 h, production of (R)-3-hydroxybutyric acid was accumulated to 11.8 g/L.

EXAMPLE 3 Genetically Modification of Bacillus subtilis

The genetically modified Bacillus subtilis comprises an exemplary (R)-3-hydroxybutyric acid synthesis pathway as shown in FIG. 1.

Plasmid Construction

Similar to the modification of Corynebacterium glutamicum, the target gene fragment t3-OXCT1-BDH1 was digested from the cloning vector PUC57-t3-OXCT1-BDH1, and then inserted into the digested and purified plasmid pMA5 to construct recombinant vectors. For confirming the insertion, the constructed recombinant vectors were transformed into competent bacterial strain DH5a for clone selection. The presence of the inserted fragment t3-OXCT1-BDH1 was confirmed by colony-PCR using specific primers, restriction enzyme digestion and sequencing. The confirmed vector was the expression vector named as pMA5-t3-OXCT1-BDH1. FIG. 3 is the plasmid map for pMA5-t3-OXCT1-BDH1.

Generation of Genetically Modified Bacillus subtilis

The Bacillus subtilis WB600 cells were made competent. Then, the expression vector pMA5-t3-OXCT1-BDH1 was transformed into this competent cell and used for producing (R)-3-hydroxybutyric acid. The transformed bacteria were plated on LB agar plate containing 100 mg/L ampicillin for clone selection.

This modified Bacillus subtilis harboring plasmid pMA5-t3-OXCT1-BDH1 was named as Bacillus subtilis NNB01.

EXAMPLE 4 Fermentation of the Genetically Modified Bacillus subtilis

The genetically modified Bacillus subtilis NNB01 was taken out from a glycerol stock stored at −80° C. and used to inoculate a 5000 mL flask containing 500 mL seed medium (10 g/L tryptone, 10 g/L NaCl, 5 g/L Yeast extract, Ammonium sulfate 0.4 g/L, Potassium dihydrogen phosphate 0.19 g/L, Magnesium sulfate 0.16 g/L). The cells were cultured at 37° C. for 20 hours, at which time the absorbance at 600 nm (known as OD₆₀₀) reaches 0.4-0.5.

Next, the 500 mL seed culture was inoculated into a 7-liter fermenter filled with 5 liters fermentation medium, which is the same as the seed medium described above. The pH of the medium was controlled at 7.2^(˜)7.5 after autoclaving. The tank pressure was maintained at 0.05 Mpa, and the cultivation temperature was controlled at 37° C. The fermentation began with an agitation speed of 700 rpm and an aeration rate of 1.3 vvm.

At the later stage of the fermentation, the pH of the medium was controlled at 7.2. The dissolved oxygen concentration (dO₂) was controlled at 25^(˜)30% by adjusting aeration rate and agitation speed. To control residual sugar level, sugar was fed slowly while the concentration of the original sugar dropped to about 3.0% and the residual sugar was controlled at 1.5%^(˜)2.0%.

After 60 hours, the production of (R)-3-hydroxybutyric acid was accumulated to 10.2 g/L.

EXAMPLE 5 Isolation of Fermentation Broth and Extraction of (R)-3-hydroxybutyric Acid

The fermentation broth was harvested from the fermenter and centrifuged at 4500 rpm. After centrifugation, cell pellets were discarded, and the supernatant was collected and mixed with 1% diatomaceous earth. After 30 min of mixing, clear supernatant was recovered from the mixture.

Next, the clear supernatant was concentrated using a nanofiltration membrane, and further purified by passing through a 732 cation exchange resin. The pass-through containing (R)-3-hydroxybutyric acid was collected, giving 56.8 g of (R)-3-hydroxybutyric acid with a yield of 92.5%.

The purity of (R)-3-hydroxybutyric acid was determined by high performance liquid chromatography. The chromatographic column was Shim-pack Vp-ODSC18 column (150 L×4.6). The mobile phase consisted of acetonitrile: water (v/v)=15:85, UV detection wavelength was 210 nm, injection volume was 20 μL, flow rate was 1 mL/min, column temperature was 10° C. The purity of (R)-3-hydroxybutyric acid was 98% and the specific optical value was [α] D20=−25° (C=6%, H₂O).

FIG. 4 is HPLC spectrum showing the production of (R)-3-hydroxybutyric acid from the genetically modified microorganism (Top: reference, Bottom: sample).

EXAMPLE 6 Preparation of Sodium (R)-3-hydroxybutyrate

5 g of (R)-3-hydroxybutyric acid was neutralized with an equivalent amount of sodium hydroxide at 25° C. The obtained neutralized product was concentrated, filtered and dried, giving sodium (R)-3-hydroxybutyrate powder with a yield of 81%. The obtained salt has a melting point of 152° C. and a specific optical value [α]_(D) ²⁰ of −14.1° (C=10%, H2O).

In summary, the present invention disclosed a genetically modified microorganism comprising a (R)-3-hydroxybutyric acid pathway and being able of producing (R)-3-hydroxybutyric acid, and a method for producing (R)-3-hydroxybutyric acid at a high yield using the modified microorganism, which was proved to be safe and non-toxic food grade. The engineered strain and manufacturing process that were disclosed in this invention has broad industrial application prospects. 

What is claimed is:
 1. A genetically modified microorganism having (R)-3-hydroxybutyrate biosynthesis capability, comprising one or more exogenous nucleic acids encoding at least one (R)-3-hydroxybutyric acid pathway enzyme selected from succinyl-CoA transferase, acetoacetyl-CoA synthase, and 3-HB dehydrogenase.
 2. The genetically modified microorganism of claim 1, wherein the genetically modified microorganism is able to produce (R)-3-hydroxybutyric acid in a pathway comprising the following steps: (i) converting pyruvic acid and coenzyme A to acetyl-CoA, (ii) converting acetyl-CoA into acetoacetyl-CoA, (iii) converting acetoacetyl-CoA to acetoacetic acid, and (iv) converting acetoacetic acid to (R)-3-hydroxybutyric acid.
 3. The genetically modified microorganism of claim 1, wherein the exogenous nucleic acid is codon-optimized gene for expression in the genetically modified microorganism.
 4. The genetically modified microorganism of claim 1, wherein the pathway enzyme encoded by exogenous nucleic acids is succinyl-CoA transferase, acetoacetyl-CoA synthase, or 3-HB dehydrogenase.
 5. The genetically modified microorganism of claim 1, wherein the pathway enzyme encoded by exogenous nucleic acids are succinyl-CoA transferase, acetoacetyl-CoA synthase and 3-HB dehydrogenase.
 6. The genetically modified microorganism of claim 1, wherein the exogenous nucleic acid encoding succinyl-CoA transferase is at least 90% identical to the nucleotide sequence of SEQ ID NO:1.
 7. The genetically modified microorganism of claim 1, wherein the exogenous nucleic acid encoding acetoacetyl-CoA synthase is at least 90% identical to the nucleotide sequence of SEQ ID NO:2.
 8. The genetically modified microorganism of claim 1, wherein the exogenous nucleic acid encoding 3-HB dehydrogenase is at least 90% identical to the nucleotide sequence of SEQ ID NO:3.
 9. The genetically modified microorganism of claim 1, wherein the microorganism is nonpathogenic microorganism selected from the group consisting of Corynebacterium glutamicum, Bacillus subtilis, Brevibacterium lactofermentum, Brevibacterium difficile, Brevibacterium flavum, and Brevibacterium breve.
 10. The genetically modified microorganism of claim 9, wherein the microorganism is Corynebacterium glutamicum or Bacillus subtilis.
 11. The genetically modified microorganism of claim 9, wherein the microorganism is Corynebacterium glutamicum.
 12. The genetically modified microorganism of claim 11, wherein the Corynebacterium glutamicum is the the strain deposited at the China General Microbiological Culture Collection Center under the accession number CGMCC No.
 13111. 13. A method for producing (R)-3-hydroxybutyric acid, comprising using the genetically modified microorganism of claim
 1. 14. The method of claim 13, wherein the method is a one-step fermentation process comprising fermenting the genetically modified microorganism of claim
 1. 15. A recombinant nucleic acid segment encoding succinyl-CoA transferase, acetoacetyl-CoA synthase, and 3-HB dehydrogenase. 